Mechanisms of cancer cell metastasis are poorly understood and there are currently are no reliable biomarkers or treatments available for this deadly disease. This is a particular problem in pancreatic and colon cancer, where most patients already display advanced metastatic disease at the time of diagnosis. Studying the metastatic process has proven difficult, because metastatic cells represent only a rare population of cells that reside in the tumor proper and established metastatic cell lines have undergone significant genetic drift over the years due to extended passaging in culture. This has severally hindered the application and interpretation of gene and protein profiling technologies. Thus, there is a crucial need to design more physiologically relevant systems to identify true, functionally important, molecular signatures of this disease. In this regards, important recent work has shown that it is possible to isolate rare metastatic cells from various organs in vivo. These cells when placed back into animals show significantly increased ability to metastasize a second time. Using this enrichment approach and gene profiling technology, a few important metastatic signatures (e.g. Twist, RhoC) have been identified and validated functionally. However, it has been estimated that only 40-60% of genetic changes observed at the mRNA level are actually reflected at the protein level making the functional relevance of most genetic profiles of metastatic cells questionable and the selection of candidate genes for validation problematic. Thus, there is a crucial need to identify functionally relevant protein signatures that predict metastatic disease. Such proteins would not only serve as true biomarkers of metastatic cancer, but also as novel therapeutic targets. To address this important need, we will use invivo selection to enrich for pancreatic cancer cells that spontaneously metastasize to lymph nodes of mice. We will utilize the Kras/lnk4a/Arf transgenic mouse model of pancreatic cancer which closely recapitulates the human form of metastatic cancer. The protein and phosphoprptein profiles of highly metastatic cells will then be determine using large-scale quantitative proteomics for comparison to control non-metastatic cells isolated from the primary tumor site. Proteins that signify metastatic cells will then be functionally validated using a gene knockout/knockin approach and animal models of cell metastasis. Finally, our published and preliminary data indicate that the src/CAS/Crk/PEAK signaling pathway is upregulated in highly metastatic cells selected in vivo. We will mechanistically determine how this signaling pathway facilitates metastasis and determine at what step in the metastastic process it impacts using high resolution imaging of metastasizing cells in live animals. Therefore our overall goal is to identify and functionally validate novel biomarkers of metastatic cells and determine how the src/CAS/Crk/PEAK pathway contributes to pancreatic cancer progression and metastasis in vivo. The overall goal of this work is to determine how cancer cells leave the primary tumor and spread throughout the body. Work on this proposal will identify new protein markers that will aide the clinician in the diagnosis of the spread of cancer and determine how molecular signals within the cell control this process.